The metabotropic glutamate receptor, mGlu5, plays a fundamental role in many neuronal processes including synapse formation, synaptic plasticity, and changes in synaptic efficacy. Not surprisingly, impaired mGlu5 signaling is implicated in disorders of synaptogenesis such as Fragile X Syndrome (FXS), autism, and obsessive compulsive disorder (OCD). Indeed, genetically or pharmacologically blocking mGlu5 function robustly improves animal models of these disorders; however exploratory clinical trials have exhibited varying degrees of success. Rather than invalidating mGlu5 as a therapeutic target, such results highlight the need for a better understanding of receptor function including its cell and location specificity. For example, we have shown that 60-90% of mGlu5 is located on intracellular membranes where it couples to distinct signaling systems versus its cell surface counterpart. Importantly, intracellular mGu5 is sufficient for establishing hippocampal and striatal long term depression, a form of synaptic learning and memory that is dysfunctional in FXS, autism and OCD. The objective of the proposed research is to develop animal models that will enable testing of both location-specific mGlu5 signaling and the ability of candidate therapeutics to affect receptors on the intracellular membranes versus the cell surface. Our central hypothesis is that the effects of signaling by mGlu5 in vivo are ?location dependent?. In Aim 1, we will take advantage of CRISPR technology to generate two mouse strains: one will incorporate a short C-terminal tag on mGlu5 that will target the receptor solely to the ER and nuclear membranes; and the other will incorporate a short N-terminal epitope on mGlu5 that will target the receptor solely to the cell surface. In Aim 2, we will use these unique animals to capitalize on our recent observation suggesting that intracellular mGlu5, but not cell-surface-localized mGlu5, is critical in synaptic models of learning and memory. We hypothesize that blocking intracellular-restricted mGlu5 will inhibit changes in synaptic plasticity underlying learning and memory as well as motor, social and anxiety-like behaviors whereas blocking cell-surface-restricted mGlu5 will not do so. This would be the first report of location-specific functions of intracellular versus cell surface-localized mGlu5 in vivo. Because mGlu5 is one of a growing number of receptors that signal from inside the cell, the proposed experiments will enhance knowledge of other intracellular receptors as well. Future in vivo studies targeting drugs to intracellular versus cell-surface-localized receptors are expected to lead to the development of new and effective therapeutic tools for FXS, autism, OCD, and other mGlu5-modulated disorders.